1. Field of the Invention
The invention relates generally to the field of biochemistry. More specifically, the invention relates to tagging of biomolecules using synthetic azido substrates.
2. Description of Related Art
Protein isoprenylation is a general term of which protein farnesylation and protein geranylgeranylation are examples. It is a type of post-translational modifications involving the covalent attachment of polyisoprenoids, for example, a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid, typically through a thioether bond to a C-terminal cysteine residue of proteins (Fu, 1999). To date, three enzymes are known to isoprenylate proteins, viz., protein farnesyltransferase (FTase) (Reiss, 1990), protein geranylgeranyltransferase type I (GGTase-I) (Seabra, 1991) and protein geranylgeranyltransferase type II (GGTase-II) (Moores, 1991). FTase utilizes farnesyl diphosphate (FPP) and selectively alkylates the cysteine residue fourth from the C-terminus in a conserved isoprenylation motif designated the “CAAX box”, where “C” is a cysteine residue, “A” as an aliphatic residue, and “X” is either S, M, Q, A, or T (single-letter amino acid codes). GGTase-I and GGTase-II are responsible for linking a geranylgeranyl group, from geranylgeranyl diphosphate (GGPP), to a cysteine residue in the C terminal CAAX (where X is L or F), CC, or CXC motifs of the proteins (Fu, 1999). Isoprenylation promotes membrane association of the target proteins and protein-protein interactions, and is essential for the function of the modified proteins (Fu, 1999; Tamanoi, 2001).
A variety of proteins are farnesylated, including the Ras superfamily G-proteins. The post-translational modification is required for the activation of Ras proteins and their transforming potential (Fu, 1999). For this reason, farnesyltransferase has been hypothesized as an anti-tumor drug target. Farnesyltransferase inhibitors (FTIs) that inhibit FTase have been developed as potential cancer therapeutic agents and a few FTI compounds are currently under clinical evaluation (End, 2001; Tamanoi, 2001).
Efficient methods for the detection and quantification of protein prenylation are needed for the analysis of the dynamics of protein prenylation. Metabolic incorporation of radio-isotope labeled farnesyl pyrophosphate (FPP) has been used to detect farnesylated proteins, but is expensive and inconvenient (Melkonian, 1999; Gibbs, 1999). Anti-farnesylation antibodies have also been developed, but they have not been widely used, largely due to limited binding affinity and specificity (Lin, 1999; Baron, 2000). Neither approach is able to adequately enrich the farnesylated proteins from a complex protein mixture.
Global profiling of farnesylated proteins under diverse cellular environments with FTI treatments would reveal those farnesylated proteins with a change in farnesylation modification, and would reveal likely protein targets for several FTIs currently under clinical trials. This would allow characterization of the dynamics of farnesylated proteins in response to changes of cellular environment. Proteomics analysis is usually performed by 2D-gel/mass spectrometry- (Hanash, 2003) or ICAT/mass spectrometry-based proteomic methods (Gygi, 1999; Aebersold, 2003), which is typically limited to a few thousand of the most abundant proteins (Aebersold, 2003). Due to their low-to-medium abundant expression, farnesylated proteins are usually not detected, and, therefore, not quantified by these methods when whole-cell protein lysates are used as starting materials. Thus, efficient proteomic analyses of these proteins require an enrichment technology that is able to remove non-farnesylated proteins and reduces the complexity of the protein mixture. Unfortunately, such a method has not previously existed. There is, therefore, a great need in the art for techniques that may be used for purification or enrichment of farnesylated and similarly modified proteins.